Antigen

Antigen Class

TFs and others

Antigen

Setd1a

Cell type

Cell type Class

Neural

Cell type

Prefrontal Cortex

MeSH Description

The rostral part of the frontal lobe, bounded by the inferior precentral fissure in humans, which receives projection fibers from the MEDIODORSAL NUCLEUS OF THE THALAMUS. The prefrontal cortex receives afferent fibers from numerous structures of the DIENCEPHALON; MESENCEPHALON; and LIMBIC SYSTEM as well as cortical afferents of visual, auditory, and somatic origin.

Sample

source_name

Prefrontal cortex

tissue

Prefrontal cortex

age

6 weeks old

Sex

male

chip antibody

A300-289, Bethyl

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Whole prefrontal cortex was dissected from 6-week old wild-type (WT) mice. Dissected tissue was thoroughly minced on ice using razor blades, then fixed with EGS for 30 min and/or for 5 min at room temperature with methanol-free formaldehyde (Pierce, Thermo Fisher Scientific) diluted to 1% in PBS. Fixation was quenched by adding 1/10th volume of 1.25 M glycine. Fixed tissue was collected by centrifugation at 200 g for 5 min at 4ºC, then washed twice with cold PBS. Washed tissue was flash-frozen with liquid nitrogen then cryofractured using a Covaris CryoPrep Impactor on power setting 6. Nuclei and chromatin immunoprecipitation were performed as in Markenscoff-Papadimitriou et al.(Markenscoff-Papadimitriou et al., 2014): briefly, the cryofractured tissue was lysed in ChIP Lysis Buffer (50 mM Tris-HCl pH 7.5, 150 nM NaCl, 0.5% NP-40, 0.25% Sodium Deoxychoalate, 0.1% SDS) in rotation for 40 min at 4ºC. Nuclei were centrifuged (1700x g, 5 min, 4ºC), and the pellet was resuspended in shearing buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8, 0.25% SDS), then sheared on a Covaris S2 sonicator (12 or 16 min, 2% Duty Cycle, Intensity 3, 200 cycles per burst, frequency sweeping). Sheared chromatin was centrifuged (10,000 g for 10 min at 4ºC) to remove insoluble material. Sheared chromatin was diluted 5-fold with ChIP Dilution Buffer (CDB: 16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS) and pre-cleared for two hours at 4ºC with protein G dynabeads (Thermo Fisher Scientific). Each ChIP was set up with the appropriate antibody (10 mg of anti-Setd1a, 1 mg of anti-H3K27ac, -H3K4me2 and -H3K4me3, 3 mg of anti-H3K4me1, 5 mg of Mef2) and 10-20 mg of cleared chromatin, incubating then overnight at 4ºC. Protein G dynabeads were blocked overnight with 2 mg/ml yeast tRNA (Thermo Fisher Scientific), then added to antibody bound chromatin and rotated for 3 hr at 4ºC. Bead-bound chromatin was washed 5 times with LiCl Wash Buffer (100 mM Tris-HCl pH 7.5, 500 mM LiCl, 1% NP-40, 1% Sodium Deoxycholate) and once with TE (pH7.5). DNA was eluted from beads by incubating at 65ºC for 30 min with 25 mL ChIP Elution Buffer (1% SDS, 0.1 M Sodium Bicarbonate). This elution was repeated and the combined elution fractions were incubated overnight at 65ºC with proteinase K (Thermo Fisher Scientific) and RNaseA (Thermo Fisher Scientific). ChIP DNA was purified 1.8X of AMPure XP beads and eluted in 30 ml of nuclease free water. Libraries were prepared with Ovation Ultralow System V2 1-16 (NuGEN technologies) according to manufacturers' recommendation.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

33577876

Reads aligned (%)

95.9

Duplicates removed (%)

64.8

Number of peaks

6633 (qval < 1E-05)

mm9

Number of total reads

33577876

Reads aligned (%)

95.7

Duplicates removed (%)

64.9

Number of peaks

6633 (qval < 1E-05)